PHOX2B polymorphisms as Hirschsprung's disease diagnostic markers and methods based thereon

Abstract

The invention relates generally to polymorphisms or mutations of the PHOX2B gene. More particularly, the invention relates to polymorphisms or mutations of the PHOX2B gene that are responsible for the disease Hirschsprung's disease (HSCR), which is a neural crest-associated developmental disorder. Specifically, the invention relates to the detection of a single base-pair polymorphism in the PHOX2B gene that is associated with HSCR. The invention also relates to methods and kits for screening for carriers of mutations of the PHOX2B gene and the diagnosis of increased risk of HSCR. The invention further relates to diagnosing predisposition or susceptibility to increased risk of developing HSCR by screening for the presence of a polymorphism associated with HSCR. The invention also relates to compositions for screening for the polymorphism and treatment choices for patients having the polymorphism of the present invention. The invention further relates to providing polymorphisms in the PHOX2B gene for forensic use and in paternity test. The invention also relates to screening assays and therapeutic and prophylactic methods.

1. A method for screening a human subject predisposed or susceptible to Hirschsprung's disease (HSCR) associated with a genetic polymorphism in the PHOX2B gene, said method comprising:
 (a) providing a biological sample from the subject; and 
 (b) testing the sample for the presence of an adenine at nucleotide position 2250 of SEQ ID NO:1, wherein the presence of the adenine in both copies of the subject's PHOX2B gene at this position indicates that the subject is genetically predisposed to HSCR. 
 
     
     
       2. A method for diagnosing a human subject with increased risk of HSCR associated with a genetic polymorphism in the PHOX2B gene, said method comprising:
 (a) providing a biological sample from the subject; and 
 (b) testing the sample for the presence of an adenine in both copies of the subject's PHOX2B gene at nucleotide position 2250 of SEQ ID NO:1, wherein the presence of the adenine at this position indicates that the subject is genetically predisposed to Hirschsprung's disease (HSCR). 
 
     
     
       3. The method of  claim 1  or  2 , wherein step (b)comprises amplifying a fragment of the PHOX2B gene comprising two or more nucleotides including a nucleotide at position 2250. 
     
     
       4. The method of  claim 3 , wherein the fragment is amplified using PCR primers comprising nucleotide sequence of SEQ ID NOS:9 and 10. 
     
     
       5. The method of  claim 1  or  2 , wherein the biological sample is selected from the group consisting of blood, saliva, amniotic fluid, and urine.